The Study of Potentially Lignocellulolytic Actinobacteria Pseudonocardia sp. AI2.

The search for microorganisms with potential for bioconversion of lignocellulose is now of immediate interest. Industrial waste is a source of various microorganisms. This paper describes results of the research of potentially lignocellulolytic actinobacteria isolated from activated sludge of the wastewater treatment plant of a pulp and paper mill located in Komi Republic (Russia). One strain of actinobacteria, AI2, was found to be sufficiently active in terms of degradation of lignocellulose-containing materials. Testing of the AI2 isolate demonstrated its ability to synthesize cellulase, dehydrogenase and protease to various extents. The AI2 strain was found capable of biosynthesizing cellulase to 5.5 U/ml. In case of solid-phase fermentation using treated softwood and hardwood sawdust, the content of main components changed most significantly in aspen sawdust: from initial concentration of 20.4% down to 15.6% for lignin, and from 50.6% down to 31.8% for cellulose. In case of liquid-phase fermentation, the content of lignin components decreased significantly in the treated aqueous medium that contained lignosulfonates: from initial concentration of 3.6 g down to 2.1g. Taxonomic study of the AI2 strain of actinobacteria confirmed that it belongs to the rare Pseudonocardia genus of actinomycetes. Based on the results of 16S rRNA sequencing, the AI2 strain is most similar to the species Pseudonocardia carboxydivorans.

Safety and efficacy of a feed additive consisting of 25-hydroxycholecalciferol (produced by Pseudonocardia autotrophicaDSM 32858) for all pigs, all poultry for fattening and ornamental birds and other poultry species (Huvepharma NV).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of a feed additive consisting of 25-hydroxycholecalciferol (produced by Pseudonocardia autotrophica DSM 32858) for all pigs, all poultry for fattening and ornamental birds and other poultry species. The production strain P. autotrophica DSM 32858 is not genetically modified however, uncertainties remain on the possible presence of its viable cells in the final product. Due to the lack of adequate safety data and uncertainty on the presence of nano particles, the FEEDAP Panel cannot conclude on the safety of the additive for the target species and the consumer. The additive was shown not to be irritant to skin or eyes and it is not a skin sensitiser. Considering the low dusting potential of the additive, the FEEDAP Panel concluded that the exposure through inhalation is unlikely. However, the FEEDAP Panel considered that uncertainties remain on genotoxicity and on the possible presence of viable cells of P. autotrophica DSM 32858 in the final product which might have an impact on the safety for the users. The use of the feed additive is considered safe for the environment. The Panel concluded that the additive has a potential to be efficacious under the proposed conditions of use.

Beneficial bacterial-Auricularia cornea interactions fostering growth enhancement identified from microbiota present in spent mushroom substrate.

Complex dynamic bacterial-fungal interactions play key roles during mushroom growth, ranging from mutualism to antagonism. These interactions convey a large influence on mushroom's mycelial and fruiting body formation during mushroom cultivation. In this study, high-throughput amplicon sequencing was conducted to investigate the structure of bacterial communities in spent mushroom substrates obtained from cultivation of two different groups of Auricularia cornea with (A) high yield and (B) low yield of fruiting body production. It was found that species richness and diversity of microbiota in group (A) samples were significantly higher than in group (B) samples. Among the identified 765 bacterial OTUs, 5 bacterial species found to exhibit high differential abundance between group (A) and group (B) were Pseudonocardia mangrovi, Luteimonas composti, Paracoccus pantotrophus, Sphingobium jiangsuense, and Microvirga massiliensis. The co-cultivation with selected bacterial strains showed that A. cornea TBRC 12900 co-cultivated with P. mangrovi TBRC-BCC 42794 promoted a high level of mycelial growth. Proteomics analysis was performed to elucidate the biological activities involved in the mutualistic association between A. cornea TBRC 12900 and P. mangrovi TBRC-BCC 42794. After co-cultivation of A. cornea TBRC 12900 and P. mangrovi TBRC-BCC 42794, 1,616 proteins were detected including 578 proteins of A. cornea origin and 1,038 proteins of P. mangrovi origin. Functional analysis and PPI network construction revealed that the high level of mycelial growth in the co-culture condition most likely resulted from concerted actions of (a) carbohydrate-active enzymes including hydrolases, glycosyltransferases, and carbohydrate esterases important for carbohydrate metabolism and cell wall generation/remodeling, (b) peptidases including cysteine-, metallo-, and serine-peptidases, (c) transporters including the ABC-type transporter superfamily, the FAT transporter family, and the VGP family, and (d) proteins with proposed roles in formation of metabolites that can act as growth-promoting molecules or those normally contain antimicrobial activity (e.g., indoles, terpenes, ß-lactones, lanthipeptides, iturins, and ectoines). The findings will provide novel insights into bacterial-fungal interactions during mycelial growth and fruiting body formation. Our results can be utilized for the selection of growth-promoting bacteria to improve the cultivation process of A. cornea with a high production yield, thus conveying potentially high socio-economic impact to mushroom agriculture.

Update of the list of QPS-recommended microbiological agents intentionally added to food or feed as notified to EFSA 16: suitability of taxonomic units notified to EFSA until March 2022.

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre-evaluation of the safety of microorganisms, intended for use in the food or feed chains, to support the work of EFSA's Scientific Panels. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge, safety concerns and occurrence of antimicrobial resistance. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this statement, no new information was found that would change the status of previously recommended QPS TUs. Of the 50 microorganisms notified to EFSA in October 2021 to March 2022 (inclusive), 41 were not evaluated: 10 filamentous fungi, 1 Enterococcus faecium, 1 Clostridium butyricum, 3 Escherichia coli and 1 Streptomyces spp. because are excluded from QPS evaluation, and 25 TUs that have already a QPS status. Nine notifications, corresponding to seven TUs were evaluated: four of these, Streptococcus salivarius, Companilactobacillus formosensis, Pseudonocardia autotrophica and Papiliotrema terrestris, being evaluated for the first time. The other three, Microbacterium foliorum, Pseudomonas fluorescens and Ensifer adhaerens were re-assessed. None of these TUs were recommended for QPS status: Ensifer adhaerens, Microbacterium foliorum, Companilactobacillus formosensis and Papiliotrema terrestris due to a limited body of knowledge, Streptococcus salivarius due to its ability to cause bacteraemia and systemic infection that results in a variety of morbidities, Pseudonocardia autotrophica due to lack of body of knowledge and uncertainty on the safety of biologically active compounds which can be produced, and Pseudomonas fluorescens due to possible safety concerns.

Pseudonocardia terrae sp. nov., an actinobacterium isolated from rice rhizosphere soil in Thailand.

A novel actinomycete, designated strain RS11V-5T, was isolated from rhizosphere soil of Oryza sativa L. collected from Roi Et Province, Thailand, and its taxonomic position was evaluated. Cells of strain RS11V-5T were Gram-stain-positive, aerobic, and non-motile. Whole-cell hydrolysates contained meso-diaminopimelic acid, arabinose, galactose, glucose and ribose. MK-8(H4) was detected as the predominant menaquinone of this strain. The polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, hydroxy-phosphatidylmethylethanolamine, hydroxy-phosphatidylethanolamine, an unidentified phospholipid, an unidentified aminolipid and an unidentified glycolipid. The major fatty acids were iso-C16 : 0, C16 : 0 and C16 : 1 ω7c/C16 : 1 ω6c. Phylogenetic analyses based on the 16S rRNA gene sequences showed that strain RS11V-5T belonged to the genus Pseudonocardia and had high 16S rRNA sequence similarity of 99.3 % to Pseudonocardia kujensis KCTC 29062T and less than 98.4 % to other members of the genus Pseudonocardia. The DNA G+C content of the strain RS11V-5T was 73.3 mol%. Strain RS11V-5T showed 46.5 % digital DNA-DNA hybridization, 92.2 % orthologous average nucleotide identity (OrthoANI), 90.2 % ANI based on blast and 92.7 % ANI based on MUMmer to P. kujensis KCTC 29062T. Based its phenotypic, genotypic, phylogenetic and chemotaxonomic characteristics, strain RS11V-5T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia terrae sp. nov. is proposed. The type strain is RS11V-5T (=TBRC 15286T=NBRC 115296T).

Enabling Efficient Genetic Manipulations in a Rare Actinomycete Pseudonocardia alni Shahu.

Pseudonocardia species are emerging as important microorganisms of global concern with unique and increasingly significant ecological roles and represent a prominent source of bioactive natural products, but genetic engineering of these organisms for biotechnological applications is greatly hindered due to the limitation of efficient genetic manipulation tools. In this regard, we report here the establishment of an efficient genetic manipulation system for a newly isolated strain, Pseudonocardia alni Shahu, based on plasmid conjugal transfer from Escherichia coli to Pseudonocardia. Conjugants were yielded upon determining the optimal ratio between the donor and recipient cells, and designed genome modifications were efficiently accomplished, including exogenous gene integration based on an integrative plasmid and chromosomal stretch removal by homologous recombination using a suicidal non-replicating vector. Collectively, this work has made the P. alni Shahu accessible for genetic engineering, and provided an important reference for developing genetic manipulation methods in other rare actinomycetes.

Genus Pseudonocardia: What we know about its biological properties, abilities and current application in biotechnology.

The genus Pseudonocardia belongs to a group of Actinomycetes, and is a member of the family Pseudonocardiacea. The members of this genus are aerobic, Gram-positive, non-motile bacteria that are commonly found in soil, plant and environment. Although this genus has a low clinical significance; however, it has an important role in biotechnology due to the production of secondary metabolites, some of which have anti-bacterial, anti-fungal and anti-tumour effects. The use of phenotypic tests, such as gelatinase activity, starch hydrolysis, catalase and oxidase tests, as well as molecular methods, such as polymerase chain reaction, are necessary for Pseudonocardia identification at the genus and species levels.

Genome analysis and plant growth promoting activity of Pseudonocardia strain DR1-2 from the root of Dendrobium christyanum Rchb.f

Aims@#The objective of this study was to analyze the genome of endophytic actinomycete associated with orchids and evaluate its plant hormone activities, including phytohormone, siderophore, ammonia production, zinc and phosphate solubilization.@*Methodology and results@#Strain DR1-2 isolated from the roots of the Thai orchid, Dendrobium christyanum Rchb.f., was closely related to Pseudonocardia alni DSM 44104T, P. antarctica DSM 44749T and P. carboxydivorans Y8T (99.93-100% similarity) based 16S rRNA gene sequence. This strain exhibited IAA production (294.10 ± 12.17 μg/mL), phosphate solubilization (2.20 ± 0.08 solubilization Index, SI), positive for siderophore production and ammonia production (36.99 ± 2.24 μg/mL). It showed a maximum IAA of 489.73 ± 8.90 μg/mL, when optimized using 0.5% Ltryptophan, pH 6 and incubated at 30 °C for 7 days. The IAA of strain enhanced the root length, shoot length, number of roots and fresh weight of rice seedlings (Oryza sativa L. cv. RD49). The draft genome of strain DR1-2 was 6,077,423 bp in 23 contigs with G+C content of 74.6%. The average nucleotide identity-Blast (ANIb) and average nucleotide identity-MUMmer (ANIm) values of strain DR1-2 and related type strains were 95.81 to 97.25% and the digital DNA-DNA hybridization (dDDH) values were 72.60 to 74.00%, respectively. Genomic analysis of strain DR1-2 revealed that the gene encodes the enzyme involved in the phytohormones biosynthesis and gene clusters involved in the biosynthesis of bioactive metabolites.@*Conclusion, significance and impact of study@#Endophytic actinomycete, Pseudonocardia strain DR1-2 from Thai orchid, D. christyanum Rchb.f., exhibited significant IAA production and affected the growth of the plant, which was the potential source of plant hormones for agricultural applications.

Pseudonocardia abyssalis sp. nov. and Pseudonocardia oceani sp. nov., two novel actinomycetes isolated from the deep Southern Ocean.

The actinomycetes strains KRD168T and KRD185T were isolated from sediments collected from the deep Southern Ocean and, in this work, they are described as representing two novel species of the genus Pseudonocardia through a polyphasic approach. Despite sharing >99 % 16S rRNA gene sequence similarity with other members of the genus, comparative genomic analysis allowed species delimitation based on average nucleotide identity and digital DNA-DNA hybridization. The KRD168T genome is characterized by a size of 6.31 Mbp and a G+C content of 73.44 mol%, while the KRD185T genome has a size of 6.82 Mbp and a G+C content of 73.98 mol%. Both strains contain meso-diaminopimelic acid as the diagnostic diamino acid, glucose as the major whole-cell sugar, MK-8(H4) as a major menaquinone and iso-branched hexadecanoic acid as a major fatty acid. Biochemical and fatty acid analyses also revealed differences between these strains and their phylogenetic neighbours, supporting their status as distinct species. The names Pseudonocardia abyssalis sp. nov. (type strain KRD168T=DSM 111918T=NCIMB 15270T) and Pseudonocardia oceani (type strain KRD185T=DSM 111919T=NCIMB 15269T) are proposed.

Competition-based screening helps to secure the evolutionary stability of a defensive microbiome.

BACKGROUND: The cuticular microbiomes of Acromyrmex leaf-cutting ants pose a conundrum in microbiome biology because they are freely colonisable, and yet the prevalence of the vertically transmitted bacteria Pseudonocardia, which contributes to the control of Escovopsis fungus garden disease, is never compromised by the secondary acquisition of other bacterial strains. Game theory suggests that competition-based screening can allow the selective recruitment of antibiotic-producing bacteria from the environment, by providing abundant resources to foment interference competition between bacterial species and by using Pseudonocardia to bias the outcome of competition in favour of antibiotic producers. RESULTS: Here, we use RNA-stable isotope probing (RNA-SIP) to confirm that Acromyrmex ants can maintain a range of microbial symbionts on their cuticle by supplying public resources. We then used RNA sequencing, bioassays, and competition experiments to show that vertically transmitted Pseudonocardia strains produce antibacterials that differentially reduce the growth rates of other microbes, ultimately biassing the bacterial competition to allow the selective establishment of secondary antibiotic-producing strains while excluding non-antibiotic-producing strains that would parasitise the symbiosis. CONCLUSIONS: Our findings are consistent with the hypothesis that competition-based screening is a plausible mechanism for maintaining the integrity of the co-adapted mutualism between the leaf-cutting ant farming symbiosis and its defensive microbiome. Our results have broader implications for explaining the stability of other complex symbioses involving horizontal acquisition.

Pseudonocardia pini sp. nov., an endophytic actinobacterium isolated from roots of the pine tree Callitris preissii.

A Gram-positive, aerobic, actinobacterial strain with rod-shaped spores, CAP47RT, which was isolated from the surface-sterilized root of a native pine tree (Callitris preissii), grown in South Australia is described. The major cellular fatty acid of this strain was iso-H-C16:1 and major menaquinone was MK-8(H4). The diagnostic diamino acid in the cell-wall peptidoglycan was identified as meso-diaminopimelic acid. These chemotaxonomic data confirmed the affiliation of strain CAP47RT to the genus Pseudonocardia. Phylogenetic evaluation based on 16S rRNA gene sequence analysis placed this strain in the family Pseudonocardiaceae, being most closely related to Pseudonocardia xishanensis JCM 17906T (98.8%), Pseudonocardia oroxyli DSM 44984T (98.7%), Pseudonocardia thailandensis CMU-NKS-70T (98.7%), and Pseudonocardia ailaonensis DSM 44979T (97.9%). The results of the polyphasic study which contain genome comparisons of ANIb, ANIm, and digital DNA-DNA hybridization revealed the differentiation of strain CAP47RT from the closest species with validated names. This strain represents a novel species and the name proposed for this microorganism is Pseudonocardia pini sp. nov., indicating the source of this actinobacterium from a pine tree. The type strain is CAP47RT (= DSM 108967T = NRRL B-65534T). Genome mining revealed that this strain contained a variety of genes encoding enzymes that can degrade hazardous chemicals.

Pseudonocardia cytotoxica sp. nov., a novel actinomycete isolated from an Arctic fjord with potential to produce cytotoxic compound.

Herein we report the isolation of a novel actinomycete, strain MCCB 268T, from the sediment sample collected from a high Arctic fjord Kongsfjorden. MCCB 268T showed greater than 97% 16S rRNA gene sequence similarity with those of Pseudonocardia konjuensis LM 157T (98.06%), Pseudonocardia soli NW8-21 (97.22%) Pseudonocardia endophytica YIM 56035 (97.08%) and Pseudonocardia nantongensis KLBMP 1282 (97.34%) showing that the strain should be assigned to the genus Pseudonocardia. DNA-DNA hybridization with Pseudonocardia konjuensis LM 157T showed only 41.5% relatedness to strain MCCB 268T. The whole genome of the strain MCCB 268T was sequenced. Whole-genome average nucleotide identity, dDDH (%) and genome tree analysis demonstrated that strain significantly differed from other Pseudonocardia species. The G + C content was 70.5 mol%. MCCB 268T exhibited in vitro cytotoxicity and through bioassay guided fractionation followed by HPLC separation a cytotoxic compound (I) was isolated. The compound (I) was identified as 1-acetyl-ß-carboline through NMR spectra and high-resolution mass spectrometry. Compound (I) showed cytotoxicity against lung cancer cell line and mode of anticancer activity was found to be through the induction of apoptosis. Based on the genotypic and phenotypic features, MCCB 268T ought to be classified as a novel species under the genus Pseudonocardia for which the name Pseudonocardia cytotoxica sp. nov. is proposed (= CCUG72333T = JCM32718T).

A new uridine derivative and a new indole derivative from the coral-associated actinomycete Pseudonocardia sp. SCSIO 11457.

A new uridine derivative 11457 A (1), and a new indole derivative 11457B (2), together with a known compound 1H-indole-2-carbaldehyde (3), were characterized from the fermentation broth of the actinomycete Pseudonocardia sp. SCSIO 11457, an isolate associated with the scleractinian coral Galaxea fascicularis. Upon detailed spectroscopic analysis, 11457 A (1) was identified as a uridine analog, and 11457B (2) was elucidated as an indole derivative 2-hydroxy-1-(1H-indol-2-yl)pentane-1,4-dione. Biological evaluation indicated that none of compounds 1-3 showed antibacterial activities against pathogenic bacteria and cytotoxic activities against human cancer cell lines.

Pseudonocardia acidicola sp. nov., a novel actinomycete isolated from peat swamp forest soil.

A novel actinobacterium, designated strain K10HN5T, was isolated from a peat soil sample collected from Kantulee peat swamp forest, Surat Thani Province, Thailand and its taxonomic position was determined using a polyphasic approach. Strain K10HN5T contained meso-diaminopimelic acid, arabinose, galactose, glucose and ribose in its whole-cell hydrolysates. The predominant menaquinone was MK-8(H4). The major fatty acids were iso-C16 : 0, iso-C15 : 0 and iso-C16 : 1H. Mycolic acids were not present. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylmethylethanolamine, hydroxyphosphatidylethanolamine, hydroxyphosphatidylmethylethanolamine and phosphatidylinositol. The 16S rRNA gene sequence analysis indicated that it was closely related to Pseudonocardia bannensis DSM 45300T (97.9 %) and Pseudonocardia xinjiangensis JCM 11839T (97.9 %). Strain K10HN5T exhibited low average nucleotide identity and digital DNA-DNA hybridization values with P. bannensis DSM 45300T (82.6, 28.7 %) and P. xinjiangensis JCM11839T (76.3, 22.2 %). The DNA G+C content of strain K10HN5T was 72.4 mol%. Based on polyphasic data, strain K10HN5T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia acidicola sp. nov. is proposed. The type strain is K10HN5T (=TBRC 10048T=NBRC 113897T).

Chemical warfare between fungus-growing ants and their pathogens.

Fungus-growing attine ants are under constant threat from fungal pathogens such as the specialized mycoparasite Escovopsis, which uses combined physical and chemical attack strategies to prey on the fungal gardens of the ants. In defence, some species assemble protective microbiomes on their exoskeletons that contain antimicrobial-producing Actinobacteria. Underlying this network of mutualistic and antagonistic interactions are an array of chemical signals. Escovopsis weberi produces the shearinine terpene-indole alkaloids, which affect ant behaviour, diketopiperazines to combat defensive bacteria, and other small molecules that inhibit the fungal cultivar. Pseudonocardia and Streptomyces mutualist bacteria produce depsipeptide and polyene macrolide antifungals active against Escovopsis spp. The ant nest metabolome is further complicated by competition between defensive bacteria, which produce antibacterials active against even closely related species.

Enhanced long-term attenuation of 1,4-dioxane in bioaugmented flow-through aquifer columns.

Long term natural attenuation of 1,4-dioxane (dioxane) and its enhanced biodegradation after bioaugmentation with Pseudonocardia dioxanivorans CB1190 were assessed using flow-through aquifer columns. Natural attenuation of dioxane was not observed even after 2 years of acclimation. However, dioxane removal was observed in the bioaugmented columns (34% when the influent was 200 µg/L and 92% for 5 mg/L). The thmA gene that encodes the tetrahydrofuran monooxygenase that initiates dioxane degradation by CB1190 was only detected at the inoculation port and persisted for months after inoculation, implying the resiliency of bioaugmentation and its potential to offer long-term enhanced biodegradation capabilities. However, due to extensive clumping and limited mobility of CB1190, the augmented catabolic potential may be restricted to the immediate vicinity of the inoculation port. Accordingly, bioaugmentation with CB1190 seems more appropriate for the establishment of biobarriers. Bioaugmentation efficiency was associated with the availability of oxygen. Aeration of the column influent to increase dissolved oxygen significantly improved dioxane removal (p < 0.05), suggesting that (for sites with oxygen-limiting conditions) bioaugmentation can benefit from engineered approaches for delivering additional oxygen.

An EDTA-resistant pyrazinamidase from non-pathogen Pseudonocardia carboxydivorans.

OBJECTIVES: To characterize a pyrazinamidase from non-pathogen Pseudonocardia carboxydivorans. RESULTS: A pyrazinamidase gene pncA encoding a 23-kDa protein PncA-Pse from P. carboxydivorans was over-expressed in Escherichia coli and characterized. This PncA-Pse can convert both pyrazinamide and nicotinamide efficiently with the optimal pH and temperature of pH 8.5 and 45 °C, respectively. Although ferrous iron and manganese were detected in PncA-Pse, the enzymatic activity is not affected by EDTA with the final concentration of 10 mM. Moreover, the enzymatic activity was not significantly affected with the addition of several metal ions, respectively. Based on the structure modeling, the 61st histidine which is associated with the metal binding, was mutated into alanine to get mutant H61A. No activity, iron and manganese were detected for H61A, which implies that PncA-Pse is a metal enzyme with resistance of the metal ion chelator EDTA, which is different from the previous reports. CONCLUSION: This is the first characterized pyrazinamidase from the genus Pseudonocardia, a non-pathogen.

Stimulated Biosynthesis of an C10-Deoxy Heptaene NPP B2 via Regulatory Genes Overexpression in Pseudonocardia autotrophica.

Polyene macrolides, such as nystatin A1, amphotericin B, and NPP A1, belong to a large family of valuable antifungal polyketide compounds that are typically produced by soil actinomycetes. Previously, NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, was generated by introducing two amino acid substitutions in the putative NADPH-binding motif of the enoyl reductase domain in module 5 of the NPP A1 polyketide synthase in Pseudonocardia autotrophica. This derivative showed superior antifungal activity to NPP A1. In this study, another novel derivative called NPP B2 was developed, which lacks a hydroxyl group at the C10 position by site-specific gene disruption of the P450 hydroxylase NppL. To stimulate the extremely low expression of the NPP B2 biosynthetic pathway genes, the 32-kb NPP-specific regulatory gene cluster was overexpressed via site-specific chromosomal integration. The extra copy of the six NPP-specific regulatory genes led to a significant increase in the NPP B2 yield from 0.19 to 7.67 mg/L, which is the highest level of NPP B2 production ever achieved by the P. autotrophica strain. Subsequent in vitro antifungal activity and toxicity studies indicated that NPP B2 exhibited similar antifungal activity but significantly lower hemolytic toxicity than NPP B1. These results suggest that an NPP biosynthetic pathway refactoring and overexpression of its pathway-specific regulatory genes is an efficient approach to stimulating the production of an extremely low-level metabolite, such as NPP B2 in a pathway-engineered rare actinomycete strain.

Pseudonocardia Symbionts of Fungus-Growing Ants and the Evolution of Defensive Secondary Metabolism.

Actinobacteria belonging to the genus Pseudonocardia have evolved a close relationship with multiple species of fungus-growing ants, where these bacteria produce diverse secondary metabolites that protect the ants and their fungal mutualists from disease. Recent research has charted the phylogenetic diversity of this symbiosis, revealing multiple instances where the ants and Pseudonocardia have formed stable relationships in which these bacteria are housed on specific regions of the ant's cuticle. Parallel chemical and genomic analyses have also revealed that symbiotic Pseudonocardia produce diverse secondary metabolites with antifungal and antibacterial bioactivities, and highlighted the importance of plasmid recombination and horizontal gene transfer for maintaining these symbiotic traits. Here, we propose a multi-level model for the evolution of Pseudonocardia and their secondary metabolites that includes symbiont transmission within and between ant colonies, and the potentially independent movement and diversification of their secondary metabolite biosynthetic genes. Because of their well-studied ecology and experimental tractability, Pseudonocardia symbionts of fungus-growing ants are an especially useful model system to understand the evolution of secondary metabolites, and also comprise a significant source of novel antibiotic and antifungal agents.

Pseudonocardia strain improvement for stimulation of the di-sugar heptaene Nystatin-like Pseudonocardia polyene B1 biosynthesis.

Pseudonocardia autotrophica was previously identified to produce a toxicity-reduced and solubility-improved disaccharide-containing anti-fungal compound belonging to the tetraene-family, Nystatin-like Pseudonocardia Polyene A1 (NPP A1). Subsequently NPP B1, a novel derivative harboring a heptaene core structure, was produced by a pathway-engineered Pseudonocardia strain through inactivation of the specific enoly reductase gene domain in the NPP biosynthetic gene cluster. Although in vitro and in vivo efficacy and toxicity studies indicate that NPP B1 is a promising lead antifungal compound, further improvement is required to increase the extremely low production yield in the pathway-engineered strain. To overcome this challenge, we performed the N-methyl-N'-nitro-N-nitrosoguanidine (NTG) iterative random mutagenesis, followed by zone-of-inhibition agar plug assay. After three rounds of the mutagenesis-and-screening protocol, the production yield of NPP B1 increased to 6.25 mg/L, which is more than an eightfold increase compared to the parental strain. The qRT-PCR analysis revealed that transcripts of the NPP B1 biosynthetic genes were increased in the mutant strain. Interestingly, an endogenous 125-kb plasmid was found to be eliminated through this mutagenesis. To further improve the NPP B1 production yield, the 32-kb NPP-specific regulatory gene cluster was cloned and overexpressed in the mutant strain. The chromosomal integration of the extra copy of the six NPP-specific regulatory genes led to an additional increase of NPP B1 yield to 31.6 mg/L, which is the highest production level of NPP B1 ever achieved by P. autotrophica strains. These results suggest that a synergistic combination of both the traditional and genetic strain improvement approaches is a very efficient strategy to stimulate the production of an extremely low-level metabolite (such as NPP B1) in a pathway-engineered rare actinomycetes strain.